PROJECT SUMMARY/ABSTRACT Multiple myeloma (MM) is a common, devastating hematological cancer with a low rate of overall survival and a need for development of new treatments. Recent genome-wide profiling of MM patient samples has revealed a globally perturbed pattern of histone methylations underlying MM pathogenesis; in particular, an enhanced level of H3K27me3 (tri-methylation of histone H3, lysine 27) correlates well with aberrant gene silencing and with MM progression to the aggressive stage. H3K27me3 is produced by Polycomb Repressive Complex 2 (PRC2), a histone methyltransferase complex that employs either EZH2 or EZH1 as an enzymatic subunit. However, direct sequencing of MM patient samples identified no mutation of EZH2, EZH1 or other PRC2 genes. The mechanisms responsible for H3K27me3 dysregulation during pathogenesis of MM remain elusive. We recently found that aberrant amplification and over-expression of PHD Finger Protein 19 (PHF19), a gene previously reported by us to encode a new regulator of PRC2, occurs frequently in MM patients and predicts a poor clinical prognosis. We also found that aberrantly expressed PHF19, as well as its interaction with PRC2, is required for tumorigenicity of MM in vitro and in vivo. Mechanistically, PHF19 dramatically enhances chromatin occupancy of PRC2, causing H3K27 hypermethylation in MM cells. Intriguingly, MM differs from many cancers by the high expression of EZH1, a less studied H3K27 methyltransferase enzyme, which significantly contributes to MM cell growth and H3K27me3 dysregulation. We hypothesize that aberrant PHF19 amplification and overexpression represents an important MM-promoting mechanism that confers aggressive tumor features and epigenetic perturbations through hyper-activation of EZH1 and EZH2 H3K27 methyl- transferase enzymes. Dissecting the molecular events and mechanisms underlying PHF19-mediated MM tumorigenicity should provide critical insights into new anti-MM strategies. Towards this goal, we will determine whether overexpression of PHF19 is necessary and sufficient to promote tumorigenesis in rigorous cancer models of MM including a patient-derived xenograft model (aim 1); we will characterize effects of PHF19 overexpression on chromatin occupancy of PRC2 and epigenetic gene deregulations in MM cells through unbiased ChIP sequencing and transcriptome profiling approaches (aim 2); and third, we will use a combined genetic (CRISPR/cas9) and pharmacological (inhibitor) approach to determine whether both EZH1 and EZH2 are crucial effectors of PHF19-mediated tumorigenesis in MM (aim 3). Significant to the proposed research is that we will evaluate anti-MM therapeutic effects of a unique small-molecule inhibitor of EZH1 and EZH2 recently discovered by our laboratories. We expect to fully delineate an important, yet unexplored oncogenic pathway in MM by functionally characterizing PHF19 with rigorous cancer models, gain a mechanistic understanding, and determine oncogenic effectors of PHF19 for potential therapeutic interventions. We will also utilize cutting-edge techniques including the CRISPR-cas9 mediated gene editing technology and a unique small-molecule inhibitor. Therefore, completion of the proposal should result in a new understanding for mechanism of MM tumorigenesis and should yield innovative, targeted therapeutics for the treatment of this deadly cancer.